| Low temperature is one of major environmental factors that affect plant growth, development and geographical distribution. Chorispora Bungeana Fisch&CA.Mey. (C.bungeana) is a representative alpine subnival plant. It mainly distributes in continental glacier nearby of central Asia with a 3800-3900m height above sea level. During life cycle, it has to often undergo drastic temperature fluctuations from 15℃to-4℃. Through morphological research, we find Chorispora Bungeana has no special morphological characteristics that help to survive under low temperature. So we deduce physiological and molecular mechanisms mainly contribute to adapting to the freezing environment. The present study is to investigate the proteome changes of Chrispora bungeana leaves under low temperature based on two dimensional electrophoresis. Many differently expressed proteins under low temperature were successfully identified in our research. A new gene, named DEAD-box RNA helicase, was cloned based on an up-regulated protein under low temperature and its functions were further explored in present research. Our research provides basic data for analyzing the molecular mechanisms of low temperature response. The detailed research results are as followings:1, we developed a most suitable system of 2-DE for separating the total proteins from Chrispora bungeana leaves and obtained the high-resolution and reproducible gel maps. About 1000±50 and 1400±50 protein spots were detected in gels of pH 3-10 and pH4-7 respectively.2, Based on comparison and statistical analysis, we found 156 protein spots had significant changes (P≤0.05). Of these 156 spots,72 spots were up-regulated under low temperature,84 spots were down-regulated under low temperature.82 proteins were successfully identified from these 156 spots by MALDI-TOF MS and Q-TOF MS. According to functional features of identified proteins, we classified them into 13 major categories, including:photosynthesis, cell rescue/defense, protein biosynthesis, folding and degradation, transcription factors and cell signal transduction, respiratory metabolism, amino acid metabolism, lipid metabolism, energy, cytoskeleton and other metabolisms. Most of identified proteins were well known low temperature responsive proteins, but some are novel low temperature induced proteins, such as DEAD box RNA helicase. These novel low temperature induced proteins provide new insights into the low temperature stress defense mechanism in plants and deserve to be further researched.3, An identified protein named DEAD-box RNA helicase, wwas up-regulated by 7 times under low temperature, which was a novel cold induced protein and was never reported in the past. Based on the information of this protein, we cloned a new gene from Chorispora Bungeana, named CbDRH. The full-length cDNA of CbDRH was 2111bp and contained an ORF (open reading frame) of 1788bp, which was predicted to encode a protein consisting of 595 amino acids. CbDRH was an orthologous protein of Arabidopsis ethylene-responsive DEAD-box RNA helicase, AtRH30, whose functions were to date largely unknown. Structurally, CbDRH possesses all the nine conserved motifs characteristic of DEAD-box protein family in its central region.4, Transcriptional expression of CbDRH was up regulated by both chilling and freezing treatment. UV-B and ABA treatment also led to the accumulation of CbDRH transcript. However, NaCl treatment down regulated the transcriptional expression level of CbDRH. Besides, CbDRH transcript was detected in all of the tissues tested (root, stem and leaf) with little tissue specificity. Subcellular localization suggested that CbDRH is localized in the nucleus. These data supported our proteomics results and also suggested that CbDRH may function in the nucleus and be involved in several cellular stress responses in Chorispora bungeana.
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